The biosynthesis of the three non-classical clans of the beta-lactam antibiotics will be investigated. Represented by the classical penicillins and cephalosporins, collectively these drugs constitute 65% of the world antibiotic market and account for >$25B/yr in economic value. They remain an important mainstay of human health and longevity. But with wide spread use has come the inevitable rise of antibiotic-resistant infections. Structural modifications have slowed these effects, but increased reliance on the newer, non-classical classes, for example, the beta-lactamase inhibitor clavulanic acid and the potent, broad-spectrum carbapenems like thienamycin, will benefit from fundamental understanding of their biosynthesis for improved, lower cost manufacture by fermentation, semi-synthesis and, as we propose, chemoenzymatic synthesis using engineered biosynthetic pathways to produce clinically-used drugs and new structures with improved characteristics. The monocyclic beta-lactams are the simplest structurally but ironically the mechanism of their formation is unknown. A series of breakthroughs in the current grant period have set the stage to finally solve this problem. This understanding will guide experiments with the biosynthetically related monobactams, distinct for their N-sulfonated beta-lactam rings that confer the increasingly important property of comparative resistance to the Class B beta-lactamases (Zn++ metalloproteases). While in the current grant period we discovered and examined in detail the ATP-dependent beta-lactam forming reactions in both clavulanic acid and carbapenem biosynthesis, the essential oxidative bicyclic ring inversion processes to the antibiotic-active stereochemistry will be determined. The four known classes of beta-lactam antibiotics arise by convergent evolution and are emblematic of remarkably varied enzyme mechanisms of impressive synthetic efficiency. Research to understand them holds great training potential for students and affords the opportunity to make valuable contributions academically and practically to their manufacture and structural alteration to overcome resistance mechanisms.